The primary objective of the proposed research is to directly define hormonal effects on the phosphorylation state of glycogen synthase, an enzyme that can be phosphorylated in vitro at several sites per subunit (Roach and Larner, 1977). Cultured skeletal muscle will be grown in medium containing 32Pj until the labeled phosphate has completely equilibrated with cellular phosphoproteins. After treatment of cells with insulin and epinephrine, (32P) glycogen synthase will be purified using immunological techniques. Different phosphorylated sites will be resolved following treatment of (32P) glycogen synthase with cyanogen bromide and trypsin (DePaoli-Roach et al, 1980). Changes in phosphate content of specific sites induced by the hormones will be determined from the differences in the radioactivity of the 32P-labeled peptides. Several different protein kinases and phosphoprotein phosphatases exhibit a fairly well defined specificity in vitro for different sites on glycogen synthase (Soderling, 1980). Therefore, knowing which phosphorylation site(s) a hormone alters provides a clue from which it may be possible to identify the kinase or phosphatase mediating the action of the hormone. It is likely that the effects of epinephrine will be mediated by cAMP-dependent protein kinase; however, the mediator of insulin action can be predicted with less certainty. It is possible that mediators of insulin action on glycogen synthase will prove to be involved in the action of the hormone on other phosphorylation-dependent cellular processes.